1. Field of the Invention
The present invention relates to a DNA molecule, vector, host and method for cold shock inducible expression and production of heterologous polypeptides.
2. Description of the Related Art
When Escherichia coli cells grown at 37° C. are transferred to a low temperature such as 15° C., a set of proteins called cold-shock proteins is transiently induced at a very high level during a growth lag period called the acclimation phase (Jones et al., 1987). These cold-shock specific proteins include: CspA, CspB, CspG, CspI, CsdA, RbfA, NusA, and PNP (for review see Phadtare et al., 1999, 2000 and 2002; Yamanaka et al., 1998). Among them, CspA has been identified as a major cold-shock protein consisting of 70 amino acid residues.
The CspA family of E. coli consists of nine homologous proteins, CspA to CspI, but among them only CspA, CspB, CspG and CspI are cold-shock inducible. Interestingly, the cspA gene is dispensable at both normal and low growth temperatures (Bae et al., 1997). None of the CspA homologues appears to be singularly responsible for cold-shock adaptation, as members of the CspA family functionally overlap each other during cold-acclimation of cells (Xia et al., 2001). Indeed, a ΔcspAΔcspBΔcspG triple deletion strain is still viable, while a ΔcspAΔcspBΔcspGΔcspE quadruple deletion strain is unable to form colonies at low temperature. Interestingly, any single gene from the nine CspA homologues except CspD has been shown to be capable of complementing the cold-sensitivity of the quadruple deletion strain (Xia et al., 2001). It has been shown that CspA is differentially regulated from CspB, CspG and CspI (Etchegaray et al., 1996 and Wang et al., 1999). High levels of CspA production are seen between 94° C. and 10° C., while CspB and CspG are produced only after temperature shifts to below 20° C., the maximum induction being at 15° C. CspI is induced between 10-15° C. It has also been shown that CspA, CspB and CspG are induced at low temperature under conditions that completely block protein synthesis (Etchegaray et al., 1999).
The cspA expression is regulated in a complex manner, that is at levels of transcription, mRNA stability and translation efficiency (for review see Phadtare et al., 2000 and Yamanaka et al., 1998). The cspA gene has an unusually long 5′ untranslated region (5′-UTR) consisting of 159 bases. Deletion analysis of the cspA 5′-UTR showed that this region is responsible for its extreme instability at 37° C. (half-life less than 12 sec), and has positive effect on mRNA stabilization at low temperature (Jiang et al., 1996 and Mitta et al., 1997). The cspA mRNA is dramatically stabilized (half-life more than 20 min) immediately following cold shock. This stabilization is transient and is lost once cells are adapted to low temperature. This in turn regulates the expression of cspA.
The cold-shock induction of cspA is quite different from the heat-shock induction, as the cspA induction does not require a specific transcription factor. Interestingly, the cspA promoter is active at 37° C., but CspA is greatly reduced as the cspA mRNA is extremely unstable at this temperature. Thus, the 5′-UTR of the cspA mRNA plays a crucial role in its cold-shock inducibility. Recently, it has been shown that CspA is produced at 37° C. during early exponential growth phase and its mRNA becomes unstable by mid- to late-exponential growth phase (Yamanaka et al., 2001 and Brandi et al., 1999). The 5′-UTR region of the cspA mRNA contains a “Cold Box” sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence (Fang et al., 1998). The laboratory of the present inventors showed that this region is essential for the normal cspA mRNA induction after cold shock, as a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, while that of the loop sequence is less important (Xia et al., 2001).
Overexpression of a mutant cspA mRNA devoid of both the initiation codon (AUG) and the coding sequence results in a severe inhibition of growth at low temperature along with a derepression of the chromosomal cspA expression. Further, the overexpressed RNA is stably associated with the 30S and 70S ribosomes. Results from the laboratory of the present inventors demonstrated that the 5′-UTR by itself had a remarkable affinity to ribosomes at low temperature. Overproduction of the 5′-UTR at 15° C. results in delayed induction of the cold-shock response and in the prolonged synthesis of not only CspA, but also CspB and CspG (Fang et al., 1998 and Jiang et al., 1996). These effects are repressed by coproduction of the 5′-UTR together with CspA. The AT-rich sequence immediately upstream of the −35 region of the cspA promoter has been shown to function as an UP element to enhance cspA transcription. Deletion of the UP element resulted in diminished activity of the cspA promoter (Goldenberg et al., 1997 and Mitta et al., 1997). Another important factor that contributes towards higher promoter activity is the presence of a TGn motif immediately upstream of the −10 region (Kumar et al., 1993). It is reported that this motif together with the −10 region constitutes the extended −10 region and the −35 region is dispensable in the presence of this region.
Importantly, the expression of cspA is also regulated at the level of translation. The preferential synthesis of cold-shock proteins during the growth lag period (the acclimation phase) suggests that their mRNAs, unlike most other cellular (non-cold-shock) mRNAs, possess a mechanism to form the translation initiation complex at low temperature without the cold-shock ribosome factors, such as RbfA and CsdA. The recent data from the laboratory of the present inventors show that there are elements within the coding sequence of CspA that enhance its translation at low temperature. mRNAs for cold-shock proteins such as CspA, CspB, CspG, CspI, CsdA and RbfA have been proposed to have an element called the downstream box (DB) in the coding region which enhances translation initiation (Mitta et al., 1997). Originally, the DB sequence was proposed to be complementary to a region in the penultimate stem of 16S rRNA and is located a few bases downstream of the initiation codon. It has been debated how DB enhances translation initiation (Etchegaray et al., 1999 and O'Connor et al., 1999) and the originally proposed mechanism by facilitating the formation of translation preinitiation complex through binding to 16S rRNA may not be the precise mechanism.
The phenomenon termed as ‘LACE’ effect (low-temperature antibiotic effect of truncated cspA expression) was observed in E. coli (Jiang et al., 1996 and Xia et al., 2001). When a truncated cspA gene is overexpressed at low temperature, cell growth is completely blocked. This has been demonstrated to be caused due to the entrapment of almost all the cellular ribosomes by the truncated cspA mRNA. This truncated mRNA is still able to form the preinitiation complex with non-adapted ribosomes at low temperature. Unambiguous demonstration of ribosome entrapping by truncated cspA mRNA has been carried out by incorporating a terminator codon either at the second (pA01S), or the eleventh (pA10S) or the 31st (pA30S) codon in the cspA gene cloned in a pUC vector (Xia et al., 2001). At 37° C., cells carrying these plasmids are perfectly normal, while upon cold-shock cells stop to grow completely. They are unable to form colonies at 15° C. and with 35S-Met, no protein was labeled. When polysome profiles of these cells were analyzed, cells expressing only the initiation codon (pA01S) contained only monosomes without any polysome peaks. Cells with pA10S showed di- and monosomes and cells with pA30S showed tri-, di- and monosomes again without any large polysomal peaks. Furthermore, a major cellular mRNA (lpp) was shown to be excluded from polysomes by taken over being the truncated cspA mRNA. These results clearly demonstrate that the robust translatability of the cspA mRNA is determined at the step of initiation. In this study, the laboratory of the present inventors also showed that the upstream region within the 5′-UTR of the cspA mRNA plays an important role in the formation of the translation initiation complex leading to the LACE effect (Xia et al., 2001).
CspA and its homologues are proposed to be RNA chaperones by destabilizing secondary structures in mRNAs (Bae et al., 2000; Jiang et al., 1997 and Phadtare et al., 2001). Since the ΔcspAΔcspBΔcspGΔcspE quadruple deletion strain can not grow at low temperature while a single csp gene is able to complement the cold-sensitivity (Xia et al., 2001), the RNA chaperone function is considered to be crucial for efficient translation of cellular mRNAs at low temperatures by blocking stable secondary-structure formation, which is inhibitory to the mRNA translation (Phadtare et al., 2001).
Cold-inducible vectors containing the cspA promoter have been shown to be useful for expression of aggregation-prone proteins such as, preS2-S′-β-galactosidase and TolAI-β-lactamase (Mujacic et al., 1999; Vasina et al., 1997 and Vasina et al., 1996). U.S. Pat. No. 6,333,191 B1 discloses promoters of cspA and cspB and vectors carrying such promoters.
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